O-sulfated gangliosides and lyso-ganglioside derivatives

ABSTRACT

Disclosed are derivatives of gangliosides and of N-acyl-N-lyso-gangliosides, of N&#39;-acyl-N&#39;-lyso-gangliosides and N,N&#39;-di- or poly-acyl-N,N&#39;-di-lyso-gangliosides, in which at least one of the hydroxyl groups in the saccharide, sialic or ceramide residues is esterified with sulfuric acid, functional derivatives thereof, and their salts with either inorganic or organic bases and the acid addition salts, except persulfated derivatives at the hydroxyl, sialic and ceramide groups of the GM 1 , GD 1a , GD 1b  and GT 1b  gangliosides. Such compounds have antineurotoxic and neurotogenic activity and may therefore be used in pharmaceutical preparations. Also a marked modulating effect on the expression of the CD 4  molecule in immune system cells has been shown.

This application is based on PCT International Application NumberPCT/US94/01965 filed Mar. 4, 1994.

OBJECT OF THE INVENTION

The present invention relates to novel derivatives of gangliosides andof N-acyl-N-lyso-gangliosides, of N'-acyl-N'-lyso-gangliosides andN,N'-di- or poly-acyl-N,N'-dilyso-gangliosides, whereof at least one ofthe hydroxyl groups of the saccharide, sialic or ceramide residues isesterified with sulfuric acid, the functional derivatives thereof, andtheir salts with either inorganic or organic bases and the acid additionsalts, except persulfated derivatives at their hydroxyl groups in thesaccharide, sialic and ceramide moieties of the gangliosides GM₁,GD_(1a), GD_(1b), and GT_(1b).

Furthermore, the present invention concerns pharmaceutical preparationscomprising the novel derivatives of gangliosides mentioned above andtheir therapeutical application.

The novel derivatives have interesting pharmacological properties,especially protective activity against neurotoxicity induced byexcitatory amino acids, such as glutamic acid, and are thereforeforeseen to be used in therapy for the nervous system, such as forconditions following degeneration or lesions, i.e. ischemia, hypoxia,epilepsy, trauma and compression, metabolic dysfunction, aging,toxic-infective diseases and chronic neurodegeneration, such asAlzheimer's disease, Parkinson's disease or Huntington's chorea.

The novel compounds of the invention, thanks to their neuritogenicactivity, may be used advantageously in therapies aimed at nervousfunction recovery, such as in peripheral neuropathies and pathologiesassociated with neuronal damage.

Moreover, certain novel derivatives which are object of the presentinvention have valuable properties for the modulation of the expressionof specific determinants, such as CD₄, present on the surface of humancells belonging to the immune system.

The ability of the above compounds to modulate expression of the CD₄molecule, a membrane glycoprotein expressed in various cell types suchas thymocytes, lymphocytes, monocytes and macrophages, has greatapplicative potential in a wide range of human pathologies.

The novel derivatives of the present are contemplated to be usedtherapeutically in all situations wherein it is necessary to preventand/or treat infections involving CD₄₊ cells, such as in infections, theetiological agents whereof are microorganisms belonging to the humanimmunodeficiency (HIV) family of viruses.

Moreover, modulation of CD₄ is useful in systemic or organ-specificautoimmune diseases, such as multiple sclerosis, rheumatoid arthritis,chronic polyarthritis, lupus erythematosus, juvenile-onset diabetesmellitus, and also to prevent the phenomenon of organ transplantrejection as well as rejection by the transplanted material against thehost, as in the case of bone marrow transplant, and in all cases wherethe desired effect is to obtain tolerance towards "self" and "non-self"antigens.

The term "N,N'-di-lyso-gangliosides" in the aforesaid definition means aganglioside from which the natural acyl groups have been removed fromthe neuraminic nitrogen (N') and from the sphingosine nitrogen (N) thusleaving free amino groups; the word "-di" stands thus to indicate thetwo positions N and N', but not the actual number of the free aminogroups which, depending on the number of sialic acids present, can bemore than 2. The expression "N-acyl-N'-di-" or "poly-acyl" is used inthe case of synthetic analogues of gangliosides substituted in both thepositions N and N'.

The functional derivatives of the abovesaid semisynthetic gangliosideanalogues are for example the esters and amides of the carboxyl groupsof sialic acid residues, and may also be inner esters with lactone bondsbetween the sialic carboxyl groups and the hydroxyls of theoligosaccharide, analogous to those known in the case of gangliosides,and possibly also the derivatives of all these compounds, the hydroxylgroups thereof being esterified with organic acids.

Of particular interest are the metal salts of the sulfated groups of thenovel compounds and possibly also the salts of any free sialiccarboxylic groups, such as sodium, potassium, calcium, magnesium,ammonium salts. Other interesting salts are derived from organic bases,especially therapeutically acceptable bases. Also included in thepresent invention are salts deriving from metals or bases, not normallyused in therapy, and such salts may be possibly used for thepurification of the novel products. The acid-addition salts can beformed for example in derivatives where the sphingosine or neuraminicamino group is free.

The aforesaid semisynthetic ganglioside analogues are novel.

Another aspect of the present invention relates to the use of thesenovel compounds in therapy, especially to treat the abovementioneddisorders affecting the central or peripheral nervous systems or theimmune system. Still another aspect of the invention relates topharmaceutical preparations containing one or more of the novelcompounds, optionally together with a pharmaceutical excipient orvehicle.

Still another aspect of the invention relates to the use of thecompounds disclosed, i.e. the poly-sulfated derivatives at theirhydroxyl groups in the saccharide, sialic and ceramide parts of thegangliosides GM₁, GD_(1a), GD_(1b) and GT_(1b), in the therapy and inpharmaceutical preparations for the therapy of neurotoxic conditions andconditions where their neuritogenic activity can be utilized.

The acyl groups present in position N and N' of the new gangliosidederivatives can be those of the gangliosides themselves, or they can beacyl groups synthetically inserted into N,N'-dilyso-gangliosides orN-lyso-gangliosides or N'-lyso-gangliosides. The N-lyso-gangliosidederivatives are often reported in literature simply as"lyso-gangliosides" and this nomenclature is partially used also in thepresent description.

The acyl groups acylating the sphingosine (N) and/or the neuraminic (N')positions may derive from aliphatic acids and have preferably a maximumof 24 carbon atoms, especially between 12 and 16 carbon atoms and arestraightchained, or from acids having between 1 and 11 carbon atoms anda straight or branched chain, such as formic acid, acetic acid,propionic acid, butyric acids, valeric acids, especially n-valeric acid,isovaleric acid, trimethylacetic acid, caproic acid, isocaproic acid,heptanoic acid, caprylic acid, pelargonic acid, capric acid andundecanoic acid, di-tert-butyl-acetic acid and 2-propyl-valeric acid, aswell as lauric, myristic, palmitic, oleic, elaidic, and stearic acid,eicosancarbonic acid and docosanoic acid. The acyl radicals may alsoderive from aliphatic acids substituted with one or more polar units,such as halogens, in particular chlorine, bromine and fluorine, free oresterified hydroxyl groups, ketone, ketal and acetal groups derived fromaliphatic or lower araliphatic alcohols, ketoxy or aldoxy or hydrazonegroups, free or esterified mercapto groups with a lower aliphatic oraraliphatic acid or etherified with lower aliphatic or araliphaticalcohols, free or esterified carboxyl groups, free or esterifiedsulfonic groups with lower aliphatic or araliphatic alcohols, sulfamidegroups substituted with lower alkyl or aralkyl groups, sulfoxide orsulfone groups derived from lower alkyl or lower alkyl groups, nitrilgroups, free or substituted amino groups, and ammonium derivatives ofsuch amino groups.

In the present specification, the term "lower" means groups with amaximum of 6 carbon atoms unless otherwise indicated.

Other acyl radicals acylating one or both of the sphingosine andneuraminic amino groups of the new derivatives may also be those of anaromatic, araliphatic, cycloaliphatic, aliphatic-cycloaliphatic orheterocyclic acid.

Aromatic acyl groups are mainly those deriving from benzoic acid or itshomologues wherein the phenyl residue is substituted with, for example,1 to 3 C₁₋₄ alkyl or alkoxy groups, especially methyl and methoxygroups, and/or with one of the polar groups mentioned as substituents inthe aliphatic acyl radical, for example free or alkylated amino groups.

Araliphatic acyl groups have preferably a C₂₋₄ alkylene chain as thealiphatic portion, and the aromatic portion is preferably one of theabove defined aromatic groups. Cycloaliphatic acyl radicals arepreferably those deriving from an alicyclic hydrocarbon having between 3to 6 carbon atoms in the ring, such as cyclopropane, cyclobutane,cyclopentane and cyclohexane. Heterocyclic radicals derive preferablyfrom a monocyclic heterocyclic compound with just one heteroatom link,such as --O--, --N═, --NH--, or --S-- and may be aromatic or aliphaticby nature, such as acids of the pyridine group, for example nicotinic orisonicotinic acid of the furane group, such as 2-furoic acid, or of thethiophene group, such as 3-thiophene acetic acid, the imidazole group,such as the 4-imidazole-acetic acid, or of the pyrrole group, such as1-methyl-2-pyrrole-carboxylic acid.

Functional derivatives of the novel semisynthetic gangliosidederivatives according to the present invention are esters, inner estersand amides of the sialic carboxyl groups. The ester groups areparticularly derived from aliphatic alcohols and especially from thosewith a maximum of 12 and preferably 6 carbon atoms, or from araliphaticalcohols with preferably one single benzene ring, optionally substitutedwith 1-3 C₁₋₄ alkyl groups, for example methyl groups, and with amaximum of 4 carbon atoms in the aliphatic chain, or from alicyclic oraliphatic-alicyclic alcohols with one single cycloaliphatic ring and amaximum of 14 carbon atoms, or from heterocyclic alcohols with a maximumof 12 carbon atoms and preferably 6, and one single heterocyclic ringcontaining a heteroatom link chosen from the group formed by --N═,--NH--, --O--, and --S--.

The amido groups of the carboxyl functions derive from ammonia or fromamines of any class, preferably with a maximum of 12 carbon atoms.Special mention should be made of lower aliphatic amines, such asmethylamine, ethylamine, propylamine, and butylamine.

Said esterifying alcohols and amidating amines can then be substituted,especially with functions chosen from the groups formed by hydroxyl,amino, alkoxyl groups with a maximum of 4 carbon atoms, carboxyl orcarbalkoxyl with a maximum of 4 atoms in the alkyl residue, alkylaminoor dialkylamino with a maximum of 4 carbon atoms in the alkyl moieties,and they may be saturated or unsaturated, especially with one doublebond. The alcohols may be monovalent or polyvalent, particularlybivalent. Of the aliphatic alcohols, special mention should be made ofthe lower alcohols with a maximum of 6 carbon atoms, such as methylalcohol, ethyl alcohol, propyl alcohol and isopropyl alcohol, n-butylalcohol, isobutyl alcohol, tert-butyl alcohol and, of the bivalentalcohols, ethylene glycol and propylene glycol. Of the araliphaticalcohols, special mention should be made of those with just one benzeneresidue, such as benzyl alcohol and phenethyl alcohol; of the alicyclicalcohols, preference is given to those with only one cycloaliphaticring, such as cyclohexyl alcohol (cyclohexanol) or terpene alcohols.Among the heterocyclic alcohols, special mention should be made oftetrafuranol and tetrapyranol.

In the partially sulfated derivatives, i.e. derivatives in which not allof the hydroxyl groups have been sulfated, according to the presentinvention, the other hydroxyl groups can in turn be esterified withorganic aliphatic, aromatic, araliphatic or heterocyclic acids, whichacids can be the same as those defined as acylating the above-mentionedN and N' amino groups. The partial sulfuric esters of the presentinvention represent generally mixtures of different position isomers.

The sulfated compounds are easily converted into their metal or organicbase salts, for example into their alkali metal salts, especially sodiumsalts, by treatment with bases or basic salts, for example, in the caseof sodium salts, with sodium carbonate. Especially for therapeuticapplications, the sulfate esters are used in the form of such salts,especially sodium salts.

Among the most important basic gangliosides to be used in thepreparation of the novel derivatives, mention can be made of for examplethose wherein the oligosaccharide is formed by a maximum of 4 saccharideunits, and wherein the saccharide portion is unitary. It is preferableto choose hexoses from the group formed by N-acetylglucosamine andN-acetylgalactosamine. The gangliosides of said group are for exampleextracted from vertebrate brains, such as those described in the article"Gangliosides of the Nervous System" in "Glycolipid Methodology", LloydA., Witting Fd., American Oil Chemists Society, Champaign, III, 187-214(1976) (see in particular FIG. 1), for example gangliosides GM₄, GM₃,GM₂, GM₁ -GlcNAC, GD₂, GD_(1a) -GalNAC, GT_(1c), G_(Q), GT₁, andparticularly those wherein the oligosaccharide contains at least oneglucose residue or one galactose residue and one N-acetylglucosamineresidue or N-acetylgalactosamine residue and above all the following:##STR1## where Glc stands for glucose, GalNAC stands forN-acetylgalactosamine, Gal stands for galactose, NANA stands forN-acetylneuraminic acid.

One group of new derivatives according to the invention is representedby the following formula (I), wherein it is clear that not all the Rsymbols can be at the same time H and with the exclusion of thepersulfated (totally sulfated) derivatives of GM₁, GD_(1a), GD_(1b),GT_(1b) gangliosides already mentioned. ##STR2## wherein R=H or SO₃ H,with the exclusion of the case where all the R=H. ##STR3## R₂=--(CH₂)_(n) --CH₃, n=12-14 R₃ =H or acyl ##STR4## the persulfatedderivatives of GM₁, GD_(1a), GD_(1b), GT_(1b) gangliosides beingexcluded.

The excluded derivatives are actually described in the paper Biochemicaland Biophysical Research Communications Vol. 175, No. 1, Feb. 28th,1991.

Of the specific compounds of particular interest, special mention shouldbe made of the monosulfated derivatives of the following gangliosidederivatives:

N-acetyl-lyso-GM₁

N-dichloroacetyl-lyso-GM₁

N-phenylacetyl-lyso-GM₁

N-propionyl-lyso-GM₁

N-trimethylacetyl-lyso-GM₁

N-trimethoxybenzoyl-lyso-GM₁

N-nicotinoyl-lyso-GM₁

N-capronyl-lyso-GM₁

N-octanoyl-lyso-GM₁

N-decanoyl-lyso-GM₁

N-undecanoyl-lyso-GM₁

N-4-chlorobenzoyl-lyso-GM₁

N-4-benzoyl-lyso-GM₁

N-2-bromoacetyl-lyso-GM₁

in the form of mixture of sulfated isomers in different positions, andthe corresponding poly-sulfated derivatives.

Of particular interest are the mono- or poly-sulfated derivativescorresponding to the aforesaid compounds, but acylated in position N',i.e. the mono- or polysulfated derivatives of

N'-acetyl-N,N'-dilyso-GM₁

N'-dichloroacetyl-N,N'-dilyso-GM₁

N'-phenylacetyl-N,N'-dilyso-GM₁

N'-propionyl-N,N'-dilyso-GM₁

N'-trimethylacetyl-N,N'-dilyso-GM₁

N'-trimethoxybenzoyl-N,N'-dilyso-GM₁

N'-nicotinoyl-N,N'-dilyso-GM₁

N'-capronyl-N,N'-dilyso-GM₁

N'-octanoyl-N,N'-dilyso-GM₁

N'-decanoyl-N,N'-dilyso-GM₁

N'-undecanoyl-N,N'-dilyso-GM₁

N'-4-chlorobenzoyl-N,N'-dilyso-GM₁

N'-4-benzoyl-N,N'-dilyso-GM₁

N'-2-bromoacetyl-N,N'-dilyso-GM₁

in the form of mixture of sulfated isomers in different positions.

Another interesting group of compounds is the mono- or polysulfatedderivatives of N,N'-diacyl-N,N'-dilyso-gangliosides deriving from GM₁,such as

N,N'-diacetyl-N,N'-dilyso-GM₁

N,N'-di(dichloroacetyl)-N,N'-dilyso-GM₁

N,N'-di(phenylacetyl)-N,N'-dilyso-GM₁

N,N'-di(propionyl)-N,N'-dilyso-GM₁

N,N'-di(trimethylacetyl)-N,N'-dilyso-GM₁

N,N'-di(trimethoxybenzoyl)-N,N'-dilyso-GM₁

N,N'-di(nicotinoyl)-N,N'-dilyso-GM₁

N,N'-di(capronyl)-N,N'-dilyso-GM₁

N,N'-di(octanoyl) -N,N'-dilyso-GM₁

N,N'-di(decanoyl)-N,N'-dilyso-GM₁

N,N'-di(undecanoyl)-N,N'-dilyso-GM₁

N,N'-di(4-chlorobenzoyl)-N,N'-dilyso-GM₁

N,N'-di(4-benzoyl)-N,N'-dilyso-GM₁

N,N'-di(2-bromoacetyl)-N,N'-dilyso-GM₁

in the form of mixtures of sulfated isomers in different positions.

As concerns gangliosides, special mention should be made of the mono-and poly-sulfated ganglioside derivatives as emphasized above, and thusmono- and poly-sulfated derivatives of gangliosides GM₁, GD_(1a),GD_(1b), GT_(1b), GM₂, GM₃, GM₄, GM₁ -glcNAC, GD₂, GD_(1a) -GalNAC,GT_(1c), G_(Q), GT₁.

All these compounds can be easily converted into their metal salts,especially sodium salts, and these have particular importance inconsideration of their possible therapeutical applications.

It is well known that gangliosides are glycosphingolipids containingsialic acid with a basic saccharide structure bound to ceramide and oneor more molecules of sialic acid. The saccharide portion presents atleast a galactose or glucose and one N-acetylglucosamine orN-acetylgalactosamine.

The general structure of a ganglioside can so be represented as follows:##STR5## where all the components are bound by glucosidic bonds.

A large number of gangliosides have been identified which areparticularly abundant in the nervous tissue, especially in the cerebralone (Ando S.: Gangliosides in the nervous system. Neurochem. Int. 5,507537, 1983).

It has been widely demonstrated that gangliosides are able to enhancefunctional recovery both in the lesioned Peripheral Nervous System (PNS)and Central Nervous System (CNS), through the involvement of specificmembrane mechanisms and the interaction with trophic factors, as pointedout from studies in vitro on neuronal cultures (Ferrari F. et al.: Dev.Brain Res., 8:215-221, 1983; Doherty P. et al., J. Neurochem. 44:1259-1265, 1985; Skaper S. D. et al., Mol. Neurobiol. 3: 173-199, 1989).

Moreover, it has been shown that gangliosides are able to selectivelyact where mechanisms responsible for neurotoxicity have been activated,thus antagonizing the effects of the paroxistic and continuousstimulation of the excitatory amino acid receptors (Favaron M. et al:Gangliosides prevent glutamate and kainate neurotoxicity in primaryneuronal cultures of neonatal rat cerebellum and cortex. Proc. Natl.Acad. Sci. 85: 7351-7355, 1988)

Concerning the PNS, the effects of the ganglioside mixture have beenreported in models of traumatic (Gorio A. et al., Brain Res. 197:236241, 1980), metabolic (Norido F. et al., Exp. Neurol. 83: 221-232,1984) and toxic (Di Gregorio F. et al., Cancer Chemother. Pharmacol. 26:3136, 1990) neuropathies. Concerning the CNS, the positive effects ofrecovery induced by the monosialoganglioside GM₁ have been widelydescribed in ischemia models (Karpiak S. E. et al., CRC Critical Rev. inNeurobiology, vol. 5. Issue 3, pp. 221-237, 1990), as well as intraumatic (Toffano G. et al., Brain Res. 296: 233-239, 1984) andneurotoxic (Schneider et al., Science 256: 843-846, 1992) lesions. Suchresults have led to the clinical application of GM₁ in conditions ofischemic brain injury (Argentino C. et al., Stroke 20: 1143-1149, 1989)and in conditions of traumatic injury of the spinal cord (Geisler F. H.,N. Engl. J. Med. 324: 1829-1838, 1991).

In addition, it has been recently shown that gangliosides are involvedin the modulation of the expression of the receptors named CD₄, whichare present on the membrane of some lymphocytes and furthermore it hasbeen shown that such a modulation is associated with an inhibition ofthe proliferation of the HIV virus (Offner H. et al.: Gangliosidesinduce selective modulation of CD₄ from helper T lymphocytes. J.Immunol. 139: 3295-3305, 1987; Grassi F. et al.: Chemical residues ofganglioside molecules involved in interactions with lymphocyte surfacetargets leading to CD₄ masking and inhibition of mitogenicproliferation. Eur. J. Immunol. 20: 145-150, 1990; Chieco-Bianchi et al.CD₄ modulation and inhibition of HIV-1 infectivity induced bymonosialoganglioside GM₁ in vitro. AIDS 3:501-507, 1989).

The molecule named CD₄ is a membrane glycoprotein of 55 KDa expressed bythymocytes, by a "subset" of T lymphocytes and, at a lower density, bymonocytes/macrophages. The molecule can be divided into three portions:one extracellular that is divided into 4 domains, three of which have astructure that joins them to the superfamily of immunoglobulins, anintra-membrane portion of 21 amino acids (aa), and a intracytosolicportion of 40 basic aa.

In T lymphocytes CD₄ has at least two functions. On one hand, itinteracts with a non-polymorphic region of Class II HLA molecules, thusstabilizing the bond between T cell and the cell which expresses theantigen (secondary role). On the other hand, recent evidence have shownhow the interaction of CD₄ with its own ligand induces the activation ofa cytoplasmic tyrosine kinase (named p56 lck) that is in contact withthe intracytosolic portion of CD₄. The activation of the tyrosine kinaseand the subsequent phosphorylation of different substrates, among themthe gamma chain of CD₃, has a facilitatory role in the signaltransduction following the interaction between the antigen receptor andthe antigen itself. Hence, CD₄ has an active role in the mechanisms thatregulate the activation of T lymphocytes.

In addition to these relevant functions for the physiology of T cells,CD₄ is also the receptor utilized by HIV viruses to enter the targetcells.

The CD₄₊ T lymphocytes play a major role in immune system functioning.In the majority of cases, following contact with the antigen the firstcells responsible for any adaptative response are the CD₄₊ T cells,which, after the activation, may become in turn effectors of response.Alternatively, the activated CD₄₊ cells can help, by release ofcytokines, other cells (B cells, CD₈₊ T cells) to become effectors ofresponse. This is valid both for responses against foreign antigens(non-self) and for body antigens (self). Thus, CD₄₊ T cells areprimarily involved in several autoimmune diseases.

The possibility of modulating the CD₄₊ T cell function is relevant in awide range of human pathologies.

In different models, both in vitro and in vivo it has been shown that byblocking the CD₄ molecule with monoclonal antibodies, the function ofCD₄₊ T cells is inhibited. Such an inhibition leads in turn tounsuccessful proliferation, unsuccessful production of cytokines,unsuccessful production of antibodies, and suppression or lowering ofclinical expression of autoimmune symptoms in experimental models ofautoimmune pathology.

The pharmacological properties of the new O-sulfated derivatives,according to the present invention, can be emphasized by theexperimental studies performed on the following compounds:

O-SULFATED GM₁ (Liga 135)

O-POLYSULFATED GM₁ (Liga 161)

O-SULFATED GM₂ (Liga 181)

O-SULFATED GM₃ (LIGA 182)

Liga 135, Liga 161, Liga 181 and Liga 182 are prepared as described inExamples 1, 2, 3, and 4, respectively.

Hereinafter will be described the experimental models and the resultsobtained with some exemplary compounds which are object of the presentinvention.

1. Antineurotoxic effect of Liga 135 and 161 in vitro in cerebellargranule cells: protective effect against neurotoxicity induced byexogenous glutamate.

MATERIALS AND METHODS

Cell cultures

Primary cultures of cerebellar granule cells have been prepared from8-day-old Sprague-Dawley rats.

Neurons have been grown in 35 mm dishes for 11-13 days and kept in ahumidified environment (95% air and 5% CO₂) at 37° C. Cultures (2.5×10⁶cells/dish) were mainly constituted of granule cells (>95%) with a lowpercentage (<5%) of glial cells (Gallo V. et al.: Selective release ofglutamate from cerebellar granule cells differentiating in culture.Proc. Natl. Acad. Sci. USA 79, 7919-7923, 1982). Glial proliferation wasprevented by cytosine arabinofuranoside.

The Liga 135 and 161 derivatives have been solubilized in sterile waterat the concentration of 50 mM and dissolved at different concentrationsin Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO₃, 2.3 mMCaCl₂, 1 mM MgCl₂, 5.6 mM glucose, 4.6 mM Hepes, pH 7.4).

Concentrations from 200 μM to 5 μM have been tested.

Description of the model of neurotoxicity induced by exogenousglutamate: compound in a pretreatment paradigm.

The cell culture medium was aspirated from the dishes (and properlystored). The dishes were washed (3×2 ml) with Locke's solution, then thesolutions (1.5 ml) containing the compound to be tested were added, andincubated for 2 hrs in incubator at 37° C. (5% CO₂).

The treated cells were washed (3×2 ml) with Locke's solution +10%heat-inactivated fetal calf serum (without glutamic acid), then washed(3×2 ml) in Locke's solution in the absence of Mg²⁺. Glutamate was addedat 100 μM (1.5 ml) in Locke's solution (-Mg²⁺) or Locke's solution alone(Mg²⁺) was used (controls). The incubation with glutamate or withLocke's solution (-Mg²⁺) was performed for 60 minutes at roomtemperature (27° C.). The glutamate was then removed, the dishes werewashed with Locke's solution (+Mg²⁺) (2×2 ml), then incubated in thepresence of the initial medium (properly stored) for 24 hrs at 37° C. inincubator (5% CO₂).

At the end of the incubation the cell viability measured by using theMTT calorimetric test was evaluated (Mosmann T., Rapid calorimetricassay for cellular growth and survival: application to proliferation andcytotoxicity assays. J. Immunol. Meth. 65, 55-63, 1983 and modifiedaccording to Skaper S. D. et al.: Death of cultured hippocampalpyramidal neurons induced by pathological activation ofN-methyl-D-aspartate receptors is reduced by monosialogangliosides. J.Pharm. and Exp. Ther. 259, 1, 452-457, 1991). The data are expressed asED₅₀ (μM).

RESULTS

The obtained results (Table 1) show that both compounds have a markedantineurotoxic activity: the neuroprotective effect (around 100%) ofLiga 135 and 161 has been observed at concentrations of 50 and 25 μM,respectively.

                  TABLE 1                                                         ______________________________________                                        Antineuronotoxic effect of Liga 135 and 161 in                                cerebellar granule cells: protective effect on                                neurotoxicity induced by exogenous glutamate                                  Groups            ED.sub.50                                                   ______________________________________                                        1) glutamate + Liga 135                                                                         20 μM                                                    2) glutamate + Liga 161                                                                         10 μM                                                    ______________________________________                                    

2. Antineurotoxic effect of Liga 161, 181 and 182 in vitro in cerebellargranule cells: compound in cotreatment paradigm with glutamic acid.

MATERIALS AND METHODS

Cell cultures

Primary cultures of cerebellar granule cells have been preparedaccording to the method described in Materials and Methods of thepreceding experiment (1).

The Liga 161, 181 and 182 derivatives have been dissolved in sterilewater at a concentration of 50 mM. Thus, dilutions have been performedat different concentrations in Locke's solution (154 mM NaCl, 5.6 mMKCl, 3.6 mM NaHO₃, 2.3 mM CaCl₂, 5.6 mM glucose , 4.6 mM Hepes, pH 7.4).

Concentrations from 100 μM to 5 μM have been tested.

Description of the model of neurotoxicity induced by exogenousglutamate: compound in cotreatment paradigm with glutamic acid

The cell culture medium was aspirated from the dishes (and properlystored). The dishes were washed (3×2 ml) with Locke's solution in theabsence of Mg²⁺. Then, 1.5 ml of Locke's solution (-Mg²⁺) with andwithout 100 μM glutamate and with and without the compound to be testedwere added. The incubation period lasted 30 minutes (37° C.). Glutamateand the compound to be tested were then removed. The dishes were washedwith Locke's solution in the presence of Mg²⁺ (2×2 ml) and thenincubated in the presence of the initial medium (properly stored) for 24hours at 37° C. in an incubator (5% CO₂).

At the end of the incubation cell viability measured by means of the MTTcolorimetric test was evaluated (Mosmann T.: Rapid colorimetric assayfor cellular growth and survival: application to proliferation andcytotoxicity assays. J Immunol. Meth. 65, 55-63, 1983 and modifiedaccording to Skaper S. D. et al.: Death of cultured hippocampalpyramidal neurons induced by pathological activation ofN-methyl-D-aspartate receptors is reduced by monosialogangliosides. J.Pharm. and Exp. Ther. 259,1, 452-457, 1991). Data were expressed as ED₅₀(μM).

RESULTS

The data obtained (Table 2) show that the new derivatives have a markedantineurotoxic activity, even when administered simultaneously withglutamate (co-treatment): the neuroprotective effect reaches its maximumeffect (around 100%) at 100 μM.

                  TABLE 2                                                         ______________________________________                                        Antineurotoxic effect of Liga 161, 181 and 182                                in simultaneous treatment with exogenous glutamate in                         cerebellar granule cells (protective effect).                                 Groups            ED.sub.50                                                   ______________________________________                                        1) glutamate + Liga 161                                                                         48 μM                                                    2) glutamate + Liga 181                                                                         44 μM                                                    3) glutamate + Liga 182                                                                         58 μM                                                    ______________________________________                                    

3. Neuritogenic activity of the compounds Liga 135 and Liga 161

MATERIALS AND METHODS

Cell Cultures

Mouse neuroblastoma cells C1300, Neuro-2A clone (obtained from AmericanCell Type Culture Collection--Bethesda, Md.) have been seeded at adensity of 10,000 cells/well (24-Falcon) in tissue culture mediumcontaining Dulbecco's modified Eagle medium (DMEM, Gibco), 10%heatinactivated fetal calf serum (FCS, lot 7201 Seromed), penicillin(100 units per ml, Irvine) and L-glutamine (2 mM, Sigma) Cells have beenincubated at 37° C. for 24 hr, then the medium was withdrawn andsubstituted with 350 μl of fresh culture medium with and withoutcompounds to be tested.

Compounds under examination and their solubilization

The derivatives have been solubilized in sterile water.

For the different compounds, consecutive dilutions in tissue culturemedium (concentrations from 200 μM to 5 μM) were performed.

Parameters

Neuritogenic activity (% of neurite-bearing cells under the opticalmicroscope).

Culture dishes incubated with the tested compounds were analyzed under aphase contrast microscope (250×): 9 optical fields were chosen withprefixed coordinates and photographed. Then, the total number of cellswere counted, as well as the number of neurite-bearing cells (length atleast double of the cell diameter) in blind on every picture. Thepercentage of neurite-bearing cells was determined following thecounting of at least 100 cells, and the data were expressed by therespective ED₅₀ (μM) (Facci L. et al.: Promotion of neuritogenesis inmouse neuroblastoma cells by exogenous ganglioside GM₁. J. Neurochem.229-305, 1984).

RESULTS

The results obtained (Table 3) show that the Liga 135 and 161derivatives promote neuritogenesis in vitro. In particular, in theexperimental conditions tested, it turns out that:

the neuritogenic effect with Liga 161 is maximal at a dose of 200 μM(about 58% of the cells present very long and ramified neurites).

the neuritogenic effect with Liga 135 is maximal at a dose of 200 μM(about 54% of the cells present very long and branched neurites).

                  TABLE 3                                                         ______________________________________                                        Neuritogenic effect of Liga 135 and 161 in                                    neuroblastoma cells N2A                                                              Compounds                                                                             ED.sub.50 (μM)                                              ______________________________________                                               Liga 135                                                                              55 μM                                                              Liga 161                                                                              68 μM                                                       ______________________________________                                    

4. Effect of Liga 182 on the expression of the CD₄ molecule in Molt 3cells

Molt 3 cells (American Type Culture Collection--Rockville, Md., USA)have been utilized, human tumoral cell lines derived from an acutelymphoblastic leukemia and formed by T lymphocytes expressing CD₄ ontheir surface. Such a cell line was chosen due to the fact that itoverlaps, as regards the expression of the CD₄ molecule, human Tlymphocytes obtained from peripheral blood. 100% of Molt 3 cells expressCD₄, whereas only part of human T lymphocytes from peripheral bloodexpress CD₄ in a proportion varying from subject to subject. The Molt 3model has therefore the advantage of allowing a better experimentalreliability.

MATERIALS AND METHODS

Molt 3 cells (1×10⁶) have been incubated with different concentrationsof Liga 182 (from 1 μg/ml to 500 μg/ml) for 60 minutes at 37° C. inbuffered saline (PBS), with or without Fetal Calf Serum (FCS). Whenutilized, FCS was added at a concentration of 5 or 10 part/percentage(vol/vol). Following the incubation and the subsequent washing thepercentage of cells expressing CD₄ has been measured by flow cytofluorimetry using a monoclonal, fluoresceinated (mAb), specific for CD₄(DAKO T4, Dakopatts, Glostrup, Denmark) and a cytofluorimeter (EPICS V,Coulter Electronics, Hialeah, Fla., USA).

In Table 4 are reported the data concerning the percentage of Molt 3cells that express CD₄, following the incubation with the compound underexamination at the different concentrations utilized.

                  TABLE 4                                                         ______________________________________                                        Effect of Liga 182 on the expression of CD.sub.4 in                           Molt 3 cells                                                                  % MOLT-3 cells expressing CD.sub.4 on the surface                                          FCS (%)                                                          Compound    μg/ml                                                                             0           5    10                                        ______________________________________                                        Liga 182    0      95.5        98.9 98.9                                      Liga 182    10     0.2         --   --                                        Liga 182    50     0.5         --   --                                        Liga 182    100    0.9         0.1  53.7                                      Liga 182    200    --          0    0.1                                       Liga 182    500    --          0    0                                         ______________________________________                                    

The results reported in Table 4 show how the modulating effect of Liga182 is a function of a dose/response curve, and how increasing doses ofserum require increasing concentrations of Liga 182.

It is important to point out that, at the highest concentration ofserum, the Liga 182 compound is able to totally inhibit the expressionof CD₄.

CONCLUSIONS

The above-described results show a remarkably interestingpharmacological profile of the novel compounds, which are object of thepresent invention. Special mention should be made of the antineurotoxiceffect on CNS cells, and the modulatory effect on the expression of theCD₄ molecule in the immune system cells.

In consideration of the antineurotoxic effect, the novel derivatives ofthe neuraminic acid may be used in disorders associated with anexcitatory activity of the excitatory amino acids. It has beendemonstrated that such amino acids, e.g., the glutamic or aspartic acid,besides their major role in different physiological processes, e.g.synaptogenesis and neuronal plasticity, are involved in the etiogenesisand/or evolution of different disorders with neuronal dysfunctionsand/or death. Even though neuronal damage may have different causes, theneuronal dysfunctions trigger a cascade of cellular events, such as theactivation of enzymatic reactions depending on Ca⁺² ions, the influenceof Ca⁺² ions, the activation of secondary messengers, which result inneuronal death. Damage to the CNS caused by excitatory amino acidsappear for instance in ischemia, epilepsy, trauma, compression,metabolic dysfunctions, aging, toxic-infective disorders, as well as inchronic neurodegenerative disorders, such as Alzheimer's disease orHuntington's chorea (Engelsen B., Acta Neurol. Scand. "Neurotransmitterglutamate: its clinical importance", 186, 4, 337-355; Olney J. W., Annu.Rev. Pharmacol. Toxicol., "Excitatotoxic amino acids andneuropsychiatric disorders", 1990, 30, 47-71).

In addition, the novel compounds which are object of the presentinvention, in consideration of their neurite-promoting activity, may beused with advantage in the therapies aiming at nerve function recoveryin those pathologies associated with a neuronal damage, such asperipheral neuropathies.

Moreover, the capacity of such compounds to modulate the expression ofthe CD₄ molecule on immune cell surface, may be of great relevance in agreat range of human pathologies, e.g., those situations in which it isnecessary to prevent and/or treat infections in which CD₄ cells areinvolved (especially infections the etiological agents thereof aremicroorganisms belonging to the HIV virus family). Furthermore, CD₄modulation is useful in systemic autoimmune or organ-specific diseases,such as multiple sclerosis, rheumatoid arthritis, chronic polyarthritis,lupus erythematosus, juvenile-onset diabetes mellitus and in order toprevent the phenomenon of organ transplant rejection as well asrejection by the transplanted material against the host, as in the caseof bone marrow transplant, and in all cases where the desired effect isto obtain tolerance towards "self" and "non-self" antigens.

The present invention does not include derivatives such as totallypersulfated GM₁, GD_(1a), GD_(1b), and GT_(1b), whereof the modulatoryeffect of CD₄ and the proliferation inhibition of the HIV-1 virus arealready known, whereas the present invention includes the therapeuticaluse of the aforesaid compounds in consideration of their antineurotoxicand neuritogenic activity, both in direct therapeutical procedures andin the preparation and use of pharmaceutical compositions comprisingsaid compounds.

The present invention includes also a process for the preparation of thenovel compounds. Such a process involves conventional andwell-documented approaches for the esterification with sulfuric acid ofthe hydroxyl groups. Thus, the process for the preparation of novelcompounds consists in treating a ganglioside, aN-acyl-N,N'-dilyso-ganglioside, a N'-acyl-N,N'-dilyso-ganglioside, or aN,N'- or polyacyl-N,N'-dilyso-ganglioside with sulfuric acid or itsreactive derivative and, optionally, in the conversion of sialiccarboxylic groups or free hydroxyl groups into their functionalderivatives and optionally converting the obtained compounds into theirmetal salts or salts deriving from organic bases, or into their saltswith acids.

The process also involves those modifications in which the process isinterrupted during any phase and, if desired, the remaining steps areperformed later, or in which the process starts from an intermediate andthe remaining steps are performed or in which an "in situ" intermediateis formed.

The lyso-gangliosides may be prepared from gangliosides by alkalinehydrolysis, for example with tetra-alkylammonium hydroxides, sodiumhydroxide or others.

The preparation of N- or N'-mono or poly-acyl-derivatives fromN,N'-dilyso-gangliosides is described in literature.

Compounds having an acyl group on the neuraminic nitrogen can beprepared by various methods. It is possible, for example, to start withdilyso-gangliosides and then effect an intermediate provisionalprotection of the sphingosine amino group, which can be done for exampleby hydrophobic interaction with phosphatidylcholine, or by acylationwith suitable protective groups, subsequent acylation on the neuraminicnitrogen with a derivative of the acid which is to be introduced intothis position, and then deprotection on the sphingosine nitrogen.Lastly, dilyso-gangliosides can be acylated on the two amino groups withthe same acid and the diacyl compound can be exposed to the action ofenzymes which are able to selectively remove the acylamino groups fromthe sphingosine nitrogen, for example enzymes used to obtainlyso-gangliosides from gangliosides, such as theglycosphingolipid-ceramide-deacylase enzyme (see plan 1).N-monoacyl-N,N'-dilyso-gangliosides can however also be obtained bydeacylating N,N'-diacyl-N,N'-dilyso-gangliosides on the neuraminicnitrogen by selective chemical hydrolysis, for example with 0.1 molaralcoholic potassium hydroxide.

The procedure for the preparation of N-acyl-N,N'-dilyso-gangliosides,and N'-acyl-N,N'-dilyso-gangliosides andN,N'-diacyl-N,N'-dilyso-gangliosides, comprises acylatingN,N'-dilyso-gangliosides with the acids corresponding to the acyl groupsto be introduced, optionally followed by selectively deacylatingsuitable N,N'-diacyl-N,N'-dilyso-gangliosides on the sphingosine orneuraminic nitrogen.

It is also possible to acylate N-acyl-N,N'-dilyso-gangliosides orN'-acyl-N,N'-dilyso-gangliosides with acids corresponding to the acylgroup to be introduced, for the preparation of N,N'-diacylatedderivatives in which the two acyl groups may be different.

N-acylation according to the aforesaid procedure can be effected in theconventional manner, for example by reacting the starting products withan acylating agent, especially with a functional derivative of the acid,the residue of which is to be introduced. Thus, it is possible to use ahalogen or an anhydride as the functional derivative of the acid, andthe acylation is carried out preferably in the presence of a tertiarybase, such as pyridine or collidine. Anhydrous conditions can be used atroom temperature or at higher temperatures, or the Schotten-Baumannmethod can also be used to advantage, operating in aqueous conditions inthe presence of an organic base. In some cases it is also possible touse esters of the acids as reactive functional derivatives. To acylate,it is also possible to use methods with activated carboxy derivatives,such as those used in peptide chemistry, for example the method usingmixed anhydrides or derivatives obtainable with carbodiimide derivativesor isoxazole salts. Of all the preparation methods, the following arethe most appropriate:

1. reaction of the lyso-ganglioside derivative with the azide of theacid;

2. reaction of the lyso-ganglioside derivative with an acylimidazole ofthe acid obtainable from the acid with N,N'-carbonyldiimidazole;

3. reaction of the lyso-ganglioside derivative with a mixed anhydride ofthe acid and of trifluoroacetic acid;

4. reaction of the lyso-ganglioside derivative with the chloride of theacid;

5. reaction of the lyso-ganglioside derivative with the acid in thepresence of a carbodiimide (such as dicyclohexylcarbodiimide) andoptionally a substance such as 1-hydroxybenzotriazole;

6. reaction of the lyso-ganglioside derivative with the acid by heating;

7. reaction of the lyso-ganglioside derivative with a methyl ester ofthe acid at a high temperature;

8. reaction of the lyso-ganglioside derivative with a phenol ester ofthe acid, for example an ester with para-nitrophenol;

9. reaction of the lyso-ganglioside derivative with an ester derivedfrom the exchange between a salt of the acid and1-methyl-2-chloropyridinium iodide.

It has already been explained how it is possible to obtain selectivepartial acylation both on the sphingosine and on the neuraminicnitrogen. Scheme 1 illustrates these procedures.

Enzymatic deacylation of N,N'-diacyl-N,N'-dilyso-gangliosides on thesphingosine nitrogen as previously reported can be effected under thesame conditions as those used for the partial deacylation ofgangliosides, for example as described in J. Biochem., 103, 1 (1988).The double deacylation of N,N'-diacyl-N,N'-dilyso-gangliosides toN,N'-dilyso-gangliosides can be effected in the same way as thepreparation of de-N-acetyl-lyso-gangliosides as described for example inBiochemistry 24, 525 (1985); J. Biol. Chem. 255, 7657, (1980); Biol.Chem. Hoppe Seyler 367, 241, (1986): Carbohydr. Research 179, 393(1988); Bioch. Bioph. Res. Comn. 147, 127 (1987).

The aforesaid publication in Carbohydr. Research 179 also describes amethod for selective deacylation on the neuraminic nitrogen by theaction of KOH (0.1M) in 90% normal butanol with the ganglioside GM₃.This type of deacylation reaction can be applied toN,N'-diacyl-N,N'-dilyso-gangliosides to obtainN-acyl-N,N'-dilyso-gangliosides. ##STR6##

The preferred method for the esterification of hydroxyl groups accordingto the process of the present invention is performed preferably with areactive derivative of sulfuric acid, preferably by treatment with asulfur trioxide/dimethylformamide complex in the presence of a base,such as triethylamine, or with sulfur trioxide/trimethylamine indimethylformamide complex, then with trifluoroacetic acid indichloromethane (Compare Biochemical and Biophysical ResearchCommunications, Vol. 175, No. 1, Feb. 28th, 1991). With such methods,which can vary according to temperature condition, solvents used andduration of the reaction, it is possible to obtain partial sulfuricesters of the hydroxyl groups or total esters, hence persulfatedcompounds.

The functional modification to be eventually performed, if desired, onthe compounds obtained from the conversion with the said derivatives ofsulfuric acid, is also done according to well-known methods, thusexcluding those methods which could affect the basic gangliosidestructure, such as involving highly acid agents, or which would beanyway performed in critical alkaline or acid conditions, or even thosemethods which would bring forth to an unwanted alkylation of thehydroxyl groups of the saccharide, sialic or ceramide portion.

The esterification of the sialic carboxyl groups or their conversioninto amides can be performed for example as described in the U.S. Pat.No. 4,713,374 of 15 Dec. 1987.

Amides can be prepared for example according to the following methods:

a) reaction of the carboxyl esters with ammonia or amines;

b) reaction of derivatives according to the invention with carboxylgroups activated with ammonia or amines.

Acylation of the hydroxyl groups on the saccharide, sialic and ceramideportion can be performed for example by means of an acid halide or anacid oxide, preferably in the presence of a tertiary base.

Another aspect of the present invention is directed to pharmaceuticalpreparations including, as active ingredients, one or more of the novelcompounds and, in particular, those above emphasized. Such preparationscan be formulated for oral, rectal, parenteral, local or transdermaladministration, thus being in a solid or semisolid form, for examplepills, tablets, gelatine capsules, capsules, suppositories, softgelatine capsules. For parenteral use predetermined forms forintramuscular or transdermal administration or suitable for infusions orintravenous injection may be used, and they can therefore be prepared assolutions of the active ingredients, or as lyophilized forms of theactive ingredients to be mixed with one or more excipients orpharmaceutically acceptable solvents, suitable to these uses andosmolarity-compatible with the physiological fluids.

For local administration preparations in the form of sprays, for examplenasal sprays, creams and ointments for topical use or bandagesadequately prepared for transdermal administration are considered.

The preparations of the present invention can be administered both tohumans and animals. The preparations contain preferably 0.01% to 0.1% ofthe active ingredient in the case of solutions, sprays, ointments andcreams, and 1% to 100%--preferably 5% to 50%--of the active ingredientin the case of solid preparations. Dosage depends on the indication, thedesired effect, and preferred route of administration.

The present invention also concerns the therapeutic use of the novelsemisynthetic analogues for the above-said indications. The daily doseto be administered in humans by parenteral route (subcutaneous orintramuscular), or by transdermal or oral route, is generally between0.5 and 5 mg of the active ingredient per kg of body weight. In thepreparations reported hereinafter a dose of 150 mg per unit can bereached. The daily dose may be divided in two or more partial doses tobe administered at intervals.

The following examples illustrate the preparation of the novelsemisynthetic analogues which are object of the present invention, aswell as the preparations that contain them as active ingredients.

EXAMPLE 1

O-SULFATED GM₁ (Liga 135)

500 mg (0.31 mmoles) of GM₁ are dissolved in 5 ml of anhydrousdimethylformamide. 0.44 ml (3.2 mmoles) of triethylamine and 245 mg (1.6mmoles) of sulfur trioxide/dimethylformamide complex are then added.

React under stirring at room temperature for 5 hours, then precipitatein 10 volumes of acetone. Dissolve the raw compound in 50 ml of 1% Na₂CO₃, dialyze against H₂ O and then lyophilize.

Yielded product: 480 mg.

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, the product shows a R_(f) between 0.3 and 0.4. Molar ratio ofsulfate/neuraminic acid groups 1/1 (determination of sulfate groups byion chromatography and determination of neuraminic acid using theresorcinol method).

Characteristic absorption I.R. S═O groups: 1260 cm⁻¹ (KBr).

EXAMPLE 2

O-POLYSULFATED GM₁ (Liga 161)

500 mg (0.32 mmoles) of GM₁ are dissolved in 5 ml of anhydrousdimethylformamide. 0.88 ml (6.37 mmoles) of triethylamine and 976 mg(6.37 mmoles) of a sulfur trioxide/dimethylformamide complex are thenadded.

React under stirring at room temperature for 5 hours, then precipitatein 10 volumes of acetone. Dissolve the raw compound in 50 ml of 1% Na₂CO₃, dialyze against H₂ O and then lyophilize.

Yielded product: 650 mg.

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, the product shows a R_(f) between 0.01 and 0.05. Molar ratio ofsulfate/neuraminic acid groups 5/1 (determination of sulfate groups byion chromatography and determination of neuraminic acid using theresorcinol method).

Characteristic absorption I.R. S═O groups: 1260 cm⁻¹ (KBr).

EXAMPLE 3

O-SULFATED-GM₂ (Liga 181)

100 mg (0.07 mmoles) of GM₂ are dissolved in 1 ml of anhydrousdimethylformamide. Then 100 μl (0.7 mmoles) of triethylamine and 54 mg(0.35 mmoles) of sulfur trioxide/dimethylformamide complex are added.

React under stirring at room temperature for 5 hours, then precipitatein 10 volumes of acetone. Dissolve the raw product in 10 ml of 1% Na₂CO₃, dialyze against H₂ O and then lyophilize.

Yielded product: 126 mg.

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, the product shows a R_(f) between 0.01 and 0.1. Molar ratio ofthe sulfate/neuraminic acid groups 4/1 (determination of sulfate groupsby ion chromatography and determination of neuraminic acid using theresorcinol method).

Characteristic absorption I.R. groups S═O: 1260 cm⁻¹ (KBr).

EXAMPLE 4

O-SULFATED GM₃ (Liga 182)

100 mg (0.08 mmoles) of GM₃ are dissolved in 1 ml of anhydrousdimethylformamide. Then, 0.11 ml (0.8 mmoles) of triethylamine and 62.4mg (0.41 mmoles) of sulfur trioxide/dimethylformamide complex are added.

React under stirring at room temperature for 5 hours, then precipitatein 10 volumes of acetone. Dissolve the raw product in 50 ml of 1% Na₂CO₃, dialyze against H₂ O and then lyophilize.

Yielded product: 116 mg.

Chromatographed. on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, the product shows a R_(f) between 0.05 and 0.15. Molar ratio ofsulfate/neuraminic acid groups 3/1 (determination of sulfate groups byion chromatography and determination of neuraminic acid using theresorcinol method).

Characteristic absorption I.R. S═O groups: 1260 cm⁻¹ (KBr).

EXAMPLE 5

O-SULFATED N-ACETYL-LYSO GM₁

500 mg (0.37 mmoles) of N-acetyl-lyso GM₁ are dissolved into 5 ml ofanhydrous dimethylformamide. Then, 0.5 ml (3.7 mmoles) of triethylamineand 283 mg (1.85 mmoles) of the sulfur trioxide/dimethylformamidecomplex are added.

React under stirring at room temperature for 5 hours, then precipitatein 10 volumes of acetone. The raw product is dissolved in 50 ml of 1%Na₂ CO₃, dialyzed against H₂ O and then lyophilized.

Yielded product: 500 mg.

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, the product shows a R_(f) between 0.15 and 0.28. Molar ratio ofsulfate/neuraminic acid groups 1/1 (determination of sulfate groups byion chromatography and determination of neuraminic acid using theresorcinol method).

Characteristic absorption I.R. S═O groups: 1260 cm⁻¹ (KBr).

EXAMPLE 6

O-SULFATED N-DICHLOROACETYL-LYSO GM₁

500 mg (0.35 mmoles) of N-dichloroacetyl-lyso GM₁ are dissolved into 5ml of anhydrous dimethylformamide. Then, 0.48 ml (3.5 mmoles) oftriethylamine and 268 mg (1.75 mmoles) of the sulfurtrioxide/dimethylformamide complex are added.

React under stirring at room temperature for 5 hours, then precipitatein 10 volumes of acetone. The rough product is dissolved in 50 ml 1% Na₂CO₃, dialyzed against H₂ O and then lyophilized.

Yielded product: 480 mg.

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, the product shows a R_(f) between 0.15 and 0.31. Molar ratio ofsulfate/neuraminic acid groups 1/1 (determination of sulfate groups byion chromatography and determination of neuraminic acid using theresorcinol method).

Characteristic absorption I.R. S═O groups: 1260 cm⁻¹ (KBr).

EXAMPLE 7

O-SULFATED N-PHENYLACETYL-LYSO GM₁

500 mg (0.35 mmoles) of N-phenylacetyl-lyso GM₁ are dissolved into 5 mlof anhydrous dimethylformamide. Then 0.48 ml (3.5 mmoles) oftriethylamine and 268 mg (1.75 mmoles) of the sulfurtrioxide/dimethylformamide complex are added.

React under stirring at room temperature for 5 hours then precipitate in10 volumes of acetone. The raw product is dissolved in 50 ml of 1% Na₂CO₃, dialyzed against H₂ O and then lyophilized.

Yielded product: 470 mg.

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, the product shows a R_(f) between 0.15 and 0.30. Molar ratio ofsulfate/neuraminic acid groups 1/1 (determination of sulfate groups byion chromatography and determination of neuraminic acid using theresorcinol method).

Characteristic absorption I.R. S═O groups: 1260 cm⁻¹ (KBr).

EXAMPLE 8

O-SULFATED N,N'-DI(DICHLOROACETYL)-DILYSO GM₁

500 mg (0.34 mmoles) of N,N'-di(dichloroacetyl)-N,N'-dilyso GM₁ aredissolved into 5 ml of anhydrous dimethylformamide. Then 0.46 ml (3.4mmoles) of triethylamine and 260 mg (1.7 mmoles) of the sulfurtrioxide/dimethylformamide complex are added.

React under stirring at room temperature for 5 hours, then precipitatein 10 volumes of acetone. The raw product is dissolved in 50 ml of 1%Na₂ CO₃, dialyzed against H₂ O and then lyophilized.

Yielded product: 465 mg.

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, the product shows a R_(f) between 0.12 and 0.25. Molar ratio ofsulfate/neuraminic acid groups 1/1 (determination of sulfate groups byion chromatography and determination of neuraminic acid using theresorcinol method).

Characteristic absorption I.R. S═O groups: 1260 cm⁻¹ (KBr).

EXAMPLE 9

O-SULFATED N'-TRIMETHOXYBENZOYL-N'-LYSO GM₁

500 mg (0.29 mmoles) of N'-trimethoxybenzoyl-N'-lyso GM₁ are dissolvedinto 5 ml of anhydrous dimethylformamide. Then 0.39 ml (2.9 mmoles) oftriethylamine and 222 mg (1.45 mmoles) of the sulfurtrioxide/dimethylformamide complex are added.

React under stirring at room temperature for 5 hours then precipitate in10 volumes of acetone. The rough product is dissolved in 50 ml of 1% Na₂CO₃, dialyzed against H₂ O and then lyophilized.

Yielded product: 455 mg.

Chromatographed on silica-gel plate with chloroform/methanol/CaCl₂ 0.3%60/35/8, the product shows a R_(f) between 0.20 and 0.35. Molar ratio ofsulfate/neuraminic acid groups 1/1 (determination of sulfate groups byion chromatography and determination of neuraminic acid using theresorcinol method).

Characteristic absorption I.R. S═O groups: 1260 cm⁻¹ (KBr).

EXAMPLE 10

INJECTABLE PHARMACEUTICAL PREPARATIONS

    ______________________________________                                        Preparation No. 1                                                             One 2-ml vial contains:                                                       Active ingredient        5 mg                                                 Sodium chloride          16 mg                                                Citrate buffer pH = 6                                                         In water for injection, q.s. to                                                                        2 ml                                                 Preparation No. 2                                                             One 2-ml vial contains:                                                       Active ingredient        50 mg                                                Sodium chloride          16 mg                                                Citrate buffer pH = 6                                                         In water for injection, q.s. to                                                                        2 ml                                                 Preparation No. 3                                                             One 4-ml vial contains:                                                       Active ingredient        100 mg                                               Sodium chloride          32 mg                                                Citrate buffer pH = 6                                                         In water for injection, q.s. to                                                                        4 ml                                                 ______________________________________                                    

EXAMPLE 11

PHARMACEUTICAL PREPARATIONS IN TWO VIALS

These preparations are prepared in two vials. The first vial containsthe active ingredient in the form of a lyophilized powder in quantitiesvarying from 10% to 90% in weight, together with a pharmaceuticallyacceptable excipient, such a glycine or mannitol. The second vialcontains a solvent, such as sodium chloride and a citrate buffer.

The contents of both vials are mixed up immediately beforeadministration and the lyophilized active ingredient is rapidlydissolved, thus resulting in an injectable solution.

    ______________________________________                                        Method No. 1                                                                  a. One 2-ml vial of lyophilized powder contains:                              Active ingredient          5 mg                                               Glycine                    30 mg                                              b. One 2-ml vial of solvent contains:                                         Sodium chloride            16 mg                                              Citrate buffer                                                                In water for injection, q.s. to                                                                          2 ml                                               Method No. 2                                                                  a. One 3-ml vial of lyophilized powder contains:                              Active ingredient          5 mg                                               Mannitol                   40 mg                                              b. One 2-ml vial of solvent contains:                                         Sodium chloride            16 mg                                              Citrate buffer                                                                In water for injection, q.s. to                                                                          2 ml                                               Method No. 3                                                                  a. One 3-ml vial of lyophilized powder contains:                              Active ingredient          50 mg                                              Glycine                    25 mg                                              b. One 3-ml vial of solvent contains:                                         Sodium chloride            24 mg                                              Citrate buffer                                                                In water for injection, q.s. to                                                                          3 ml                                               Method No. 4                                                                  a. One 3-ml vial of lyophilized powder contains:                              Active ingredient          50 mg                                              Mannitol                   20 mg                                              b. One 3-ml vial of solvent contains:                                         Sodium chloride            24 mg                                              Citrate buffer                                                                In water for injection, q.s. to                                                                          3 ml                                               Method No. 5                                                                  a. One 5-ml vial of lyophilized powder contains:                              Active ingredient          150 mg                                             Glycine                    50 mg                                              b. One 4-ml vial of solvent contains:                                         Sodium chloride            32 mg                                              Citrate buffer                                                                In water for injection, q.s. to                                                                          4 ml                                               Method No. 6                                                                  a. One 5-ml vial of lyophilized powder contains:                              Active ingredient          100 mg                                             Mannitol                   40 mg                                              b. One 4-ml vial of solvent contains:                                         Sodium chloride            32 mg                                              Citrate buffer                                                                In water for injection, q.s. to                                                                          4 ml                                               Method No. 7                                                                  a. One 3-ml vial contains:                                                    Active ingredient                                                             Micronized sterile         40 mg                                              b. One 3-ml vial of solvent contains:                                         Tween 80 ®             10 mg                                              Sodium chloride            24 mg                                              Phosphate buffer                                                              In water for injection, q.s. to                                                                          3 ml                                               Method No. 8                                                                  a. One 5-ml vial contains:                                                    Active ingredient                                                             Micronized sterile         100 mg                                             b. One 4-ml vial of solvent contains:                                         Tween 80 ®             15 mg                                              Soybean lecithin           5 mg                                               Sodium chloride            36 mg                                              Citrate buffer                                                                In water for injection, q.s. to                                                                          4 ml                                               ______________________________________                                    

EXAMPLE 12

PHARMACEUTICAL PREPARATIONS FOR TRANSDERMAL ADMINISTRATION

    ______________________________________                                        Preparation No. 1                                                             A bandage contains:                                                           Active ingredient    100       mg                                             Glycerol             1.6       g                                              Polyvinyl alcohol    200       mg                                             Polyvinyl pyrrolidone                                                                              100       mg                                             Excipient to increase transdermal                                                                  20        mg                                             penetration                                                                   Water                1.5       g                                              Preparation No. 2                                                             100 g ointment contain:                                                       Active ingredient    4.0       g                                              (In 5 g phospholipid liposomes)                                                                    4.0       g                                              Polyethylene glycol monostearate                                                                   1.5       g                                              Glycerol             1.5       g                                              Beta-oxybenzoic acid ester                                                                         125       mg                                             Water                72.9      g                                              ______________________________________                                    

EXAMPLE 13

PHARMACEUTICAL PREPARATIONS FOR ORAL ADMINISTRATION

    ______________________________________                                        Preparation No. 1                                                             A tablet contains:                                                            Active ingredient    20        mg                                             Single-crystal cellulose                                                                           150       mg                                             Lactose              20        mg                                             Starch               10        mg                                             Magnesium stearate   5         mg                                             Preparation No. 2                                                             A pill contains:                                                              Active ingredient    30        mg                                             Carboxymethyl cellulose                                                                            150       mg                                             Starch               15        mg                                             Lactose              10        mg                                             Sucrose              35        mg                                             Coloring agent       0.5       mg                                             Preparation No. 3                                                             A gelatine capsule contains:                                                  Active ingredient    40        mg                                             Lactose              100       mg                                             Gastroresistant covering                                                                           5         mg                                             Preparation No. 4                                                             A soft gelatine capsule contains:                                             Active ingredient    50        mg                                             Vegetable oil        200       mg                                             Beeswax              20        mg                                             Gelatine             150       mg                                             Glycerol             50        mg                                             Coloring agent       3         mg                                             ______________________________________                                    

This invention being thus described, it is obvious that these methodscan be modified in various ways. Such modifications are not to beconsidered as divergences from the very spirit and purposes of theinvention, and any modification that would appear evident to an expertin the field comes within the scope of the following claims:

We claim:
 1. Sulfated ester derivatives of gangliosides,N-acyl-N-lyso-gangliosides, N'-acyl-N'-lyso-gangliosides,N-acyl-N,N'-dilyso-gangliosides, N'-acyl-N,N'-dilyso-gangliosides,N,N'-di-lyso-gangliosides and poly-acyl-N,N'-di-lyso-gangliosides havingsaccharide, sialic and ceramide residues with hydroxyl groups, whereinat least one of the hydroxyl groups is esterified with sulfuric acid toform sulfate groups so that the molar ratio of sulfate groups to sialicresidues is from 1:1 to 5:1; and inorganic base, organic base and acidaddition salts thereof; and esters and amides of the sialic carboxylgroups; and inner esters between the sialic carboxyl groups andsaccharide hydroxyl groups.
 2. A mixture of sulfated ester derivativesaccording to claim 1, wherein said derivatives comprise two or moredifferent mono-sulfated positional isomers and sodium salts thereof. 3.The sulfated ester derivatives according to claim 1 having a pluralityof sulfated hydroxyl groups.
 4. The sulfated ester derivatives accordingto claim 1, which are N-acyl-N,N'-dilyso-gangliosides orN'-acyl-N,N'-dilyso-gangliosides having an acyl residue of an aliphaticacid with a maximum of 24 carbon atoms.
 5. The sulfated esterderivatives according to claim 4, wherein the acyl residue issubstituted with a polar group selected from the group consisting ofhalides, free or esterified hydroxyl groups, and free or esterifiedmercapto groups.
 6. The sulfated ester derivatives according to claim 1,which are N-acyl-N,N'-dilyso-gangliosides orN'-acyl-N,N'-dilyso-gangliosides having an acyl residue of benzoic acid,wherein said benzoic acid residue has a phenyl group which may besubstituted with (1) 1 to 3 C₁₋₄ alkyl or alkoxy groups, (2) a freeamino group or C₁₋₄ alkylamino group, or a combination of groups from(1) and (2).
 7. The derivative according to claim 1, in which the acylgroup in the N-acyl-N,N'-dilyso ganglioside orN'-acyl-N,N'-dilyso-ganglioside is an acyl moiety of an araliphatic acidwith a C₂₄ alkylene-aliphatic chain, and wherein a phenyl group may besubstituted with 1 to 3 C₁₋₄ alkyl or alkoxy groups and/or with a freeamino group or C₁₋₄ alkylamino groups.
 8. The sulfated ester derivativesaccording to claim 4, wherein the aliphatic acid is cyclopropane,cyclobutane, cyclopentane or cyclohexane carboxylic acid.
 9. Thesulfated ester derivatives according to claim 1, which areN-acyl-N,N'-dilyso-gangliosides or N'-acyl-N,N'-dilyso-gangliosideshaving an acyl residue of a heterocyclic acid with one heterocyclic ringand with one heteroatom link selected from the group consisting of--O--, --N═, --NH--, and --S--.
 10. The sulfated ester derivativesaccording to claim 1, which are a carboxylic acid ester of the sialiccarboxyl group, said carboxylic acid ester being derived from analiphatic alcohol having a maximum of 12 carbon atoms or from anaraliphatic alcohol having a benzene ring unsubstituted or substitutedwith 1 to 3 C₁₋₄ alkyl groups.
 11. The sulfated ester derivativesaccording to claim 1 which are a carboxylic amide of the sialic carboxylgroup, said carboxylic amide being derived from ammonia or from analiphatic amine with a maximum of 12 carbon atoms.
 12. Compounds ofclaim 1 having the formula ##STR7## wherein R is hydrogen or SO₃ H andR₁ is hydrogen, or ##STR8## wherein R₂ is --(CH₂)_(n) --CH₃, n is 12-14,R₃ is hydrogen or acyl, and R₄ is hydrogen, or ##STR9## with the provisothat not all R groups can be hydrogen.
 13. Sodium salts of the compoundsaccording to claim
 12. 14. A compound having a sulfated hydroxyl groupselected from the group consisting of mono-sulfated derivativesof:N-acetyl-lyso-GM₁, N-dichloroacetyl-lyso-GM₁,N-phenylacetyl-lyso-GM₁, N-propionyl-lyso-GM₁,N-trimethylacetyl-lyso-GM₁, N-trimethoxybenzoyl-lyso-GM₁,N-nicotinoyl-lyso-GM₁, N-capronyl-lyso-GM₁, N-octanoyl-lyso-GM₁,N-decanoyl-lyso-GM₁, N-undecanoyl-lyso-GM₁, N-4-chlorobenzoyl-lyso-GM₁,N-4-benzoyl-lyso-GM₁, and N-2-bromoacetyl-lyso-GM₁.
 15. A compoundhaving one or more sulfated hydroxyl groups selected from the groupconsisting of mono- and poly-sulfated derivativesof:N'-acetyl-N'-lyso-GM₁, N'-dichloroacetyl-N'-lyso-GM₁,N'-phenylacetyl-N'-lyso-GM₁, N'-propionyl-N'-lyso-GM₁,N'-trimethylacetyl-N'-lyso-GM₁, N'-trimethoxybenzoyl-N'-lyso-GM₁,N'-nicotinoyl-N'-lyso-GM₁, N'-capronyl-N'-lyso-GM₁,N'-octanoyl-N'-lyso-GM₁, N'-decanoyl-N'-lyso-GM₁,N'undecanoyl-N'-lyso-GM₁, N'-4-chlorobenizoyl-N'-lyso-GM₁,N'-4-benzoyl-lyso-GM₁, and N'-2-bromoacetyl-lyso-GM₁, wherein the molarratio of sulfate groups to sialic residues is from 1:1 to 5:1.
 16. Acompound having one or more sulfated hydroxyl groups selected from thegroup consisting of mono- and polysulfated derivativesofN,N'-diacetyl-N,N'-dilyso-GM₁,N,N'-di(dichloroacetyl)-N,N'-dilyso-GM₁,N,N'-di(phenylacetyl)-N,N'-dilyso-GM₁,N,N'-di(propionyl)-N,N'-dilyso-GM₁,N,N'-di(trimethylacetyl)-N,N'-dilyso-GM₁,N,N'-di(trimethoxybenzoyl)-N,N'-dilyso-GM₁,N,N'-di(nicotinoyl)-N,N'-dilyso-GM₁, N,N'-di(capronyl)-N,N'-dilyso-GM₁,N,N'-di(octanoyl)-N,N'-dilyso-GM₁, N,N'-di(decanoyl) -N,N'-dilyso-GM₁,N,N'-di(undecanoyl)-N,N'-dilyso-GM₁,N,N'-di(4-chlorobenzoyl)-N,N'-dilyso-GM₁,N,N'-di(4-benzoyl)-N,N'-dilyso-GM₁, andN,N'-di(2-bromoacetyl)-N,N'-dilyso-GM₁, wherein the molar rtio ofsulfate groups to sialic residues is from 1:1 to 5:1.
 17. Compoundshaving one or more sulfated hydroxyl groups selected from the groupconsisting of mono-sulfated and polysulfated derivatives of aganglioside selected from the group consisting of the followinggangliosides: GM₁, GD_(1a), GT_(1b), GM₂, GM₃, GM₄, GM₁ --GlcNAC, GD₂,GD_(1a) --GalNAC, GT_(1c), G_(Q), and GT₁, wherein the molar ratio ofsulfate groups to sialic residues is from 1:1 to 5:1.
 18. A method forinhibitingglutamate induced and N-methyl-D-aspartate inducedneurocytotoxicity which comprises administering at least one derivativeaccording to claim 1 to a patient in need thereof.
 19. A method forinhibiting glutamate induced and N-methyl-D-aspartate inducedneurocytotoxicity which comprises administering to a patient in needthereof sulfated GM₁, GD_(1a), GD_(1b) and GT_(1b) gangliosides havingthe hydroxyl sialic acid and ceramide groups thereof esterified withsulfuric acid, wherein the molar ratio of sulfate groups to sialicresidues is from 1:1to 5:1.
 20. A process for the preparation of acompound according to claim 1, which comprises reacting a ganglioside, aN-acyl-N-lyso-ganglioside N-acyl-N,N'-dilyso-gangliosides,N'-acyl-N,N'-dilyso-gangliosides, a N'-acyl-N'-lyso-ganglioside or aN,N'-di- or poly-acyl-N,N'-dilyso-ganglioside with sulfuric acid to forma partial sulfate ester ganglioside; and optionally converting sialiccarboxyl groups or free hydroxyl groups of said partial sulfate esterganglioside into esters thereof, amides thereof or inner esters betweenthe sialic carboxyl groups and saccharide hydroxyl groups; andoptionally converting the partial sulfate ester ganglioside into metalsalts or salts deriving from organic bases, or acid addition salts. 21.Pharmaceutical preparations containing, as the active ingredient, acompound according to claim 1, in association with a pharmaceuticallyacceptable excipient.
 22. Pharmaceutical preparations containing as theactive ingredients a compound selected from the group consistingofO-SULFATED GM₁ O-POLYSULFATED GM₁ O-SULFATED GM₂ and O-SULFATED GM₃ inassociation with a pharmaceutically acceptable excipient, wherein themolar ratio of sulfate groups to sialic residues is from 1:1 to 5:1. 23.A method for inhibiting cellular expression of the CD₄ molecule in apatient which comprises the administration of at least one of thederivatives of claim 1 to a patient in need thereof.
 24. The sulfatedester derivatives according to claim 1, which areN-acyl-N-lyso-gangliosides, N'-acyl-N'-lyso-gangliosides,N-acyl-N,N'-dilyso-gangliosides, N'-acyl-N,N'-dilyso-gangliosides,N,N'-di-lyso-gangliosides.
 25. A method for inhibiting glutamate inducedand N-methyl-D-aspartate induced neurocytotoxicity which comprisesadministering at least one compound according to claim 14 to a patientin need thereof.
 26. A method for inhibiting glutamate induced andN-methyl-D-aspartate induced neurocytotoxicity which comprisesadministering at least one compound according to claim 15 to a patientin need thereof.
 27. A method for inhibiting glutamate induced andN-methyl-D-aspartate induced neurocytotoxicity which comprisesadministering at least one compound according to claim 16 to a patientin need thereof.
 28. A method for inhibiting cellular expression of theCD₄ molecule in a patient which comprises the administration of at leastone of the compounds of claim 14 to a patient in need thereof.
 29. Amethod for inhibiting cellular expression of the CD₄ molecule in apatient which comprises the administration of at least one of thecompounds of claim 15 to a patient in need thereof.
 30. A method forinhibiting cellular expression of the CD₄ molecule in a patient whichcomprises the administration of at least one of the compounds of claim16 to a patient in need thereof.